PROJECT SUMMARY/ABSTRACT Pten is a prominent tumor suppressor whose tumor protective ability is exquisitely sensitive to alterations in level of expression or activity. This property has led to speculation that mechanisms controlling Pten expression, sta- bility, conformation, homo- and heterotypic protein interactions, localization, or catalytic activity, including post- translational modifications, are prominent targets for deregulation in human cancer. However, this concept has not been critically tested at the organismal level, mainly because of difficulty in manipulating Pten in mice due to its essential role in embryogenesis. Our long-term objective is to close this knowledge gap by the use of mouse models in which specific Pten domains or regulatory mechanisms are inactivated and to apply the information gained to develop innovative strategies for the treatment of human cancers with Pten alterations. As the next step in the pursuit of this goal, our objective here is to understand, at the physiological level, how the phosphor- ylation status of the C-tail region regulates Pten. Based on extensive preliminary studies, we hypothesize that individual C-tail serine/threonine residues differentially regulate the stability, localization, interactome and/or phosphatase activity of Pten in vivo, thereby impacting its tumor suppressive functions in both Akt-dependent and -independent fashions. We propose to test this hypothesis by pursuing two specific aims. In the first aim, we will comprehensively examine tissues and cultured cells from a core set of eight nonphosphorylatable and phos- phomimetic C-tail mutant mice for changes in Pten biological properties and functions. In the second aim, we will monitor these same mouse strains alongside cohorts of wildtype, Pten hypomorphic and Pten+/? mice for the development of spontaneous tumors, with emphasis on prostate and mammary gland lesions. Additionally, we will conduct a comparative analysis of pre-tumorous and tumorous tissues of these strains for alterations in Pten properties and functions. By completing these aims, we expect to gain insight into the properties of these C-tail mutants in physiologically relevant settings with regards to protein stability, localization, catalytic activity and binding partners, and to integrate these findings with information about the biological and tumor suppressive functions that these mutants have lost, preserved, or gained. The expected overall impact of this innovative proposal is that it will fundamentally advance our mechanistic understanding of the normal and neoplastic func- tions of the second most frequently mutated tumor suppressor gene in human cancer. This knowledge will con- ceptually advance the cancer biology field, improve our understanding of the Akt signaling pathway in normal physiology and cancer, and lay the foundation for the development of new therapeutic strategies that will improve the clinical outcome of cancer patients with alterations in Pten.